Coprococcus bacteria for use in the treatment of metabolic syndrome and inflammatory bowel diseases

ABSTRACT

The present invention relates to the preventive or curative treatment of metabolic syndrome and associated disorders, and inflammatory bowel diseases using  Coprococcus  bacteria and/or culture supernatants thereof.

FIELD OF THE INVENTION

The present invention relates to the field of medicine and in particularto compositions comprising bacterial probiotics for the preventive orcurative treatment of metabolic syndrome and associated disorders, andinflammatory bowel diseases.

BACKGROUND OF THE INVENTION

The metabolic syndrome is a cluster of the most dangerous heart attackand diabetes risk factors. This represents a major health problembecause a quarter of the world's adults have metabolic syndrome. Peoplewith metabolic syndrome are twice as likely to die from, and three timesas likely to have a heart attack or stroke compared with people withoutthe syndrome. People with metabolic syndrome have a five-fold greaterrisk of developing type 2 diabetes.

Inflammatory bowel diseases (IBD) is a general term used to identify agroup of inflammatory disorders of the gastrointestinal (GI) tractincluding Crohn's disease and ulcerative colitis. While the exact causeof IBD is not entirely understood, it is known to involve geneticpredisposition, dysbiosis of the gut microbiota and environmentalinfluences. IBD is characterized by the repeated alternating cycles ofclinical relapse and remission. In the absence of adequate treatment,IBD leads to chronic inflammation and thus to irreversible intestinaldamages.

Crohn's disease can affect any part of the GI tract but most commonlyaffects the end of the small intestine (the ileum) where it joins thebeginning of the colon. Crohn's disease may appear in “patches,”affecting some areas of the GI tract while leaving other sectionscompletely untouched. In Crohn's disease, the inflammation may extendthrough the entire thickness of the bowel wall. Ulcerative colitis islimited to the large intestine (colon) and the rectum. The inflammationoccurs only in the innermost layer of the lining of the intestine. Itusually begins in the rectum and lower colon, but may also spreadcontinuously to involve the entire colon. In some individuals, it isdifficult to determine whether their IBD is Crohn's disease orulcerative colitis. In these rare cases, people are given the diagnosisof indeterminate colitis (IC).

Some recent studies have revealed that the metabolic syndrome and IBDcan be treated or prevented using aryl hydrocarbon receptor (AhR)agonists or bacterial probiotics producing such agonists (see WO2018/065132 and WO 2017/032739). Aryl-hydrocarbon receptor (AhR) is aligand-activated nuclear receptor/transcription factor that regulatesgenes involved in toxicant metabolism and provides a major defense toenvironmental exposures. The AhR can be activated by dietary componentssuch as fats and fat derivatives, and there is evidence linking theactivated AhR to major diseases, including obesity (La Merill et al,2009, Environ Health Perspect, 117, 1414-1419).

In a previous patent application (WO 2018/065132), the inventorsobserved that animal models of metabolic syndrome (high fat diet(HFD)-induced metabolic syndrome or leptin deficient mice (ob/ob mice))are associated with a decreased AhR agonist activity of their gutmicrobiota and the administration of AhR agonist, either via apharmacological strategy or via an intestinal bacterium naturallyproducing AhR agonist, reduces the weight gain, and improves glucosetolerance, insulin sensitivity and fatty liver disease. They alsoobserved that, in human, the AhR agonist activity of the gut microbiotais inversely correlated with the metabolic syndrome. Furthermore, inanother previous patent application (WO 2017/032739), the inventorsdemonstrated that inoculation with lactobacilli that metabolizetryptophan and produce AHR ligands reduces colitis in an AHR-dependentmanner.

However, even if the link between AhR and the metabolic syndrome or IBDhas been elucidated, there is still a strong need of bacterialprobiotics exhibiting suitable features to be used in the prevention ortreatment of these pathologies.

SUMMARY OF THE INVENTION

The inventors herein identified a bacterial probiotic associated with ahigh AhR activity. Indeed, they demonstrated that Coprococcus comesbacteria and/or a culture supernatant thereof, are able to protect micefrom DSS-induced colitis, to alleviate diet-induced metabolicimpairments, and to reduce hepatic steatosis features such ascholesterol and HDL serum levels in high-fat diet (HFD) mice. All theseeffects were shown to be AhR dependent.

Accordingly, the present invention relates to a composition comprising aCoprococcus bacterium and/or a culture extract thereof exhibiting AhRagonist activity, for use in the treatment of a disease selected fromthe group consisting of metabolic syndrome and associated disorders, andinflammatory bowel diseases.

Preferably, the bacterium is a Coprococcus comes bacterium.

More preferably, the bacterium is Coprococcus comes strain depositedwith the Deutsche Sammlung von Mikroorganismen and Zellkulturen (DSMZ)under accession number DSM 33359, or a mutant thereof.

Preferably, the Coprococcus bacterium in the composition is a livingCoprococcus bacterium and/or the culture extract is a culturesupernatant. In particular, the composition may comprise a livingCoprococcus bacterium and a culture supernatant thereof, preferably aliving Coprococcus comes bacterium and a culture supernatant thereof.

Optionally, the composition may further comprise one or severaladditional bacterial probiotics capable of producing an AhR agonist andpreferably selected from the group consisting of bacteria belonging tothe genera Allobaculum, Adlercreutzia, Anaerostipes, Bifidobacterium,Propionibacterium, Bacteroides, Eubacterium, Enterococcus, Ruminococcusand Faecalibacterium, Escherichia coli, and lactic acid bacteria such asbacteria belonging to the genera Lactobacillus and Streptococcus, orselected from the group consisting of bacteria belonging to the generaAllobaculum, Adlercreutzia, Propionibacterium, Enterococcus, Escherichiacoli, Lactobacillus and Streptococcus. In particular, the compositionmay further comprise one or several additional bacterial probioticsselected from the group consisting of strains available under CNCMdeposit numbers CNCM I-5019, CNCM I-5020, CNCM I-5021, CNCM I-5022 andCNCM I-5023, Ruminococcus gnavus ATCC 29149, Lactobacillus salivariusDSM 20555, Lactobacillus reuteri DSM 20016, Lactobacillus gasseri DSM20243, Faecalibacterium prausnitzii A2-165, Escherichia coli MG1665,Anaerostipes hadrus DSM 3319, Anaerostipes caccae DSM 14662,Anaerostipes butyraticus DSM 22094 and Allobaculum stercoricanis DSM13633. Alternatively, in another embodiment, the composition does notcomprise any additional bacterium, in particular any additionalbacterial probiotic.

Preferably, the composition is to be administered by oral or rectalroute.

Preferably, the disease is selected from metabolic syndrome orassociated disorders, said disorders being preferably selected from thegroup consisting of cardiovascular diseases, insulin resistance, glucoseintolerance, type 2 diabetes, non alcoholic fatty liver disease andlipodystrophy, more preferably being selected from metabolic syndrome orassociated disorders, said disorders being selected from the groupconsisting of cardiovascular diseases, insulin resistance, glucoseintolerance, non alcoholic fatty liver disease and lipodystrophy. Thecardiovascular diseases are preferably coronary heart diseases, morepreferably heart attack or stroke.

In embodiments wherein the disease is an inflammatory bowel disease, thedisease is preferably selected from the group consisting of Crohn'sdisease, ulcerative colitis, indeterminate colitis (IC), othernoninfective gastroenteritis, enteritis, enterocolitis and colitis, andpouchitis, more preferably is selected from Crohn's disease andulcerative colitis. Preferably, the disease is not antibiotic associatedcolitis, more preferably is not Clostridium difficile associateddiarrhea.

In another aspect, the present invention also relates to a Coprococcuscomes strain deposited with the Deutsche Sammlung von Mikroorganismenand Zellkulturen (DSMZ) under accession number DSM 33359, or a mutantthereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 : C. comes supernatant protects from DSS-induced colitis. Bodyweight loss (A), DAI (B) during DSS-induced colitis in mice treated withC. comes (bacteria+supernatant, supernatant or killed bacteria). Colonlength (C) and histological score after HES staining (D-E), after 12days of DSS-induced colitis. IL-17A, IFN-γ, IL-22 and IL-10 proteinsafter MLN cells stimulation at day 12 (F). Each graph is representativeof two distinct experiments n=5-10. Statistics: Two-way ANOVA,Bonferonni post test.

FIG. 2 : C. comes protection is AhR dependent. Fold change of AhRactivity in H1L1.1c2 (A), Cyp1a and AhRR mRNA in colon and liver atsteady state after C. comes supernatant gavage during 3 weeks (B). Bodyweight loss (C) and DAI (D) after DSS-induced colitis in AhR−/− with orwithout C. comes SN gavage. Colon length (E), histological score (F) andHES staining after 9 days of colitis (G). Each graph is representativeof two distinct experiments n=5-10. Statistics: Two-way ANOVA,Bonferonni post test.

FIG. 3 : C. comes protection passes though IL-22 secretion. Fold changeof Reg3γ, Reg3β and IL-22 mRNA in colon at steady state after C. comessupernatant gavage during 3 weeks (A). Body weight loss (B) and DAI (C)after DSS-induced colitis in IL22−/− mice. Colon length (D),histological score (E) and HES staining after 9 days of colitis (F).Each graph is representative of two distinct experiments n=7-8.Statistics: Two-way ANOVA, Bonferonni post test.

FIG. 4 : Treatment with Coprococcus comes alleviates diet-inducedmetabolic impairments. (A) Body weight gain, (B) Food intake, (C) Bloodglucose level after 16h of fasting of Cony- and HFD-fed micesupplemented with C. comes or vehicle. (D) AUC (area under the curve) ofthe OGTT, (E) Blood glucose levels after 4h of fasting during the ITT,(F) AUC of the ITT.

FIG. 5 : Treatment with Coprococcus comes reduces hepatic steatosis inHFD mice. (A) Representative pictures of H&E-stained liver sections fromindicated mice. (B) Lipid area, calculated as the percent area ofinterest (A01), in the liver cross-sections of Cony- and HFD-fed micesupplemented with C. comes or vehicle.

FIG. 6 : Treatment with Coprococcus comes reduces serum features ofHFD-induced metabolic syndrome. Concentrations of (A) cholesterol and(B) HDL from the sera of indicated mice.

FIG. 7 : Supplementation of Coprococcus comes in AhR−/− mice is notsufficient to alleviate diet-induced metabolic impairments. (A) Bodyweight gain, (B) Food intake, (C) Blood glucose level after 16h offasting of Cony- and HFD-fed mice supplemented with C. comes or vehicle.(D) AUC of the OGTT, (E) Blood glucose levels after 4h of fasting duringthe ITT, (F) AUC of the ITT.

FIG. 8 : Supplementation of Coprococcus comes in AhR−/− mice has noimpact on hepatic steatosis in HFD mice. (A) Representative pictures ofH&E-stained liver sections from indicated mice. (B) Lipid area,calculated as the percent area of interest (A01), in the livercross-sections of Cony- and HFD-fed mice supplemented with C. comes orvehicle.

DETAILED DESCRIPTION OF THE INVENTION

The inventors herein identified a bacterial probiotic associated with ahigh AhR activity. Indeed, they demonstrated that Coprococcus comesbacteria and/or their culture supernatant are able to protect mice fromDSS-induced colitis, and to alleviate diet-induced metabolic impairmentsand to reduce nonalcoholic fatty liver disease (NAFLD) features such ashepatic steatosis in high-fat diet (HFD) mice. All these effects wereshown to be AhR dependent.

Accordingly, in a first aspect; the present invention relates to acomposition comprising a Coprococcus bacterium and/or a culture extractthereof exhibiting AhR agonist activity, for use in the treatment of adisease selected from the group consisting of metabolic syndrome andassociated disorders, and inflammatory bowel diseases. It also relatesto the use of a composition comprising a Coprococcus bacterium and/or aculture extract thereof exhibiting AhR agonist activity, for themanufacture of a medicament for the treatment of a disease selected fromthe group consisting of metabolic syndrome and associated disorders, andinflammatory bowel diseases. It further relates to a method for treatinga disease selected from the group consisting of metabolic syndrome andassociated disorders, and inflammatory bowel diseases, in a subject,said method comprising administering to the subject a compositioncomprising a Coprococcus bacterium and/or a culture extract thereofexhibiting AhR agonist activity.

Coprococcus (Uniprot Taxon ID: 33042) is a genus of anaerobic cocciwhich are part of the human fecal microbiota. In preferred embodiments,the Coprococcus bacterium is Coprococcus comes (e.g. Uniprot Taxon ID:410072). Coprococcus comes bacteria are commercially available, forexample from ATCC® collection (ATCC® 277581.

Coprococcus bacteria can be cultured in anaerobic conditions, at 37° C.and in a suitable medium. Examples of suitable media include, but arenot limited to, LYHBHI medium (Brain-heart infusion medium supplementedwith 0.5% yeast extract and 1% hemine) supplemented with cellobiose (1mg/ml), maltose (1 mg/ml), cysteine (0.5 mg/ml), vitamin K1 (0.0002%)and vitamin K3 (0.0002%), ATCC® Medium 1102 (Chopped meat carbohydratemedium with 0.1% cellobiose, 0.1% maltose, 0.1% starch, and 0.1% Tween80) or ATCC® Medium 260 (Trypticase soy agar/broth with defibrinatedsheep blood).

Coprococcus bacteria used in the present invention exhibit AhRactivation properties.

As used herein, the term “AhR” has its general meaning in the art andrefers to Aryl hydrocarbon receptor, a transcription factor which isactivated by diverse compounds and regulates the expression ofxenobiotic metabolism genes. Aryl hydrocarbon receptor (AhR) is a memberof the family of basic helix-loop-helix transcription factors, thebHLH-PAS (basic helix-loop-helix/Per-ARNT-Sim) family (Schmidt andBradfield, 1996, Annu Rev Cell Dev Biol. 12, 55-89; Safe S et al. 2013,Toxicol Sci., 135, 1-16). It is described in the Uniprot database underP35869. The sequences of reference in Genbank are the followings:NM_001612.1 and NP_001621.4.

The term “AhR activity” has its general meaning in the art and refers tothe biological activity associated with the activation of the AhRresulting from its signal transduction cascade, and including any of thedownstream biological effects resulting from the binding of an AhRagonist, e.g. a natural ligand, to AhR. Analyzing the AhR activationlevel may be assessed by any of a wide variety of well-known methods(Lehmann et al., 1995, Journal of Biological Chem., 270, 12953-12956; Heet al., 2011, Environ Toxicol Chem, 30, 1915-1925; and Gao et al., 2009,Anal Biochem, 393, 163-175).

The term “AhR activation properties” or “AhR agonist activity” meanspotency in being able to induce AhR activity, i.e. to activate asignaling pathway that is initiated by AhR activation, and may involveany kind of activating mechanism. Therefore, it is not always necessaryfor a microbial body itself to be an AhR ligand, and for example asecretory substance produced by a microbe may have AhR-activatingpotency. The AhR activation properties of a bacterium may be assessed bycell-based assays such as described in the examples, He et al., 2011,supra and Gao et al., 2009, supra. In particular, the AhR activationlevel may be assessed by luciferase activity in AhR-responsiverecombinant cells such as AhR-responsive recombinant guinea pig(G16L1.1c8), rat (H4L1.1c4), mouse (H1L1.1c2) and human (HG2L6.1c3)cells. The AhR activation level may also be assessed by measuring theability to stimulate AhR-dependent gene expression using recombinantmouse hepatoma (Hepa1c1c7) cell-based CALUX (H1L1.1c2 and H1L6.1c2)clonal cell lines that contain a stably integratedAhR-/dioxin-responsive element (DRE)-driven firefly luciferase plasmid(pGudLuc1.1 or pGudLuc6.1, respectively) and CAFLUX (H1G1.1c3) clonalcell lines (He et al., 2011, supra). Typically, the AhR agonist activityis measured by performing the method described in the examples, i.e. byassessing luciferase activity in AhR-responsive recombinant cells,preferably H1L1.1c2 cells, in the presence of the bacterium to be testedor a culture extract thereof, in particular a culture supernatantthereof. In particular, the AhR agonist activity may be measured byAhR-dependent chemically activated luciferase expression (CALUX)analysis in recombinant H1L1.1c2 cells containing the stably integratedDRE-driven firefly luciferase reporter plasmid pGudLuc1.1, in thepresence of the bacterium to be tested or a culture extract thereof, inparticular a culture supernatant thereof, and an increase, preferably anincrease of at least 2%, at least 5%, at least 10%, at least 20%, atleast 50%, at least 75% or at least 100%, of the luciferase (or AhR)activity by comparison to a control sample without said bacterium orculture extract indicates that the bacterium exhibits AhR agonistactivity. Preferably, the AhR agonist activity is measured by the methodof described in the examples, in particular by assessing luciferaseactivity in AhR-responsive recombinant cells, preferably H1L1.1c2 cells,and 10% culture supernatant of a Coprococcus bacterium used in theinvention increases AhR activity by at least 1.5-fold change bycomparison with AhR activity obtained in a control sample without saidculture supernatant.

The composition of the invention may comprise one or several strains ofCoprococcus, in particular one or several strains of Coprococcus comes,and/or a culture extract of one or several strains of Coprococcus, inparticular a culture extract of one or several strains of Coprococcuscomes.

Preferably, the composition of the invention comprises at least oneCoprococcus comes bacterium and/or a culture extract thereof.

More preferably, the composition of the invention comprises Coprococcuscomes bacterium deposited under accession number DSM 33359 on Nov. 21,2019 at Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH(DSMZ, Inhoffenstr. 7B, D-38124 Braunschweig, Germany) or a mutantthereof, and/or a culture extract thereof.

As used herein, the term “mutant” should be understood as a strainderived, or a strain which can be derived from a strain of the invention(or the mother strain) by means of e.g. genetic engineering, radiationand/or chemical treatment. The mutant can also be a spontaneouslyoccurring mutant. It is preferred that the mutant is a functionallyequivalent mutant, e.g. a mutant that has substantially the same, orimproved, properties (in particular AhR activation properties) as themother strain. Such a mutant is a part of the present invention.Especially, the term “mutant” refers to a strain obtained by subjectinga strain of the invention to any conventionally used mutagenizationtreatment including treatment with a chemical mutagen such as ethanemethane sulphonate (EMS) or N-methyl-N′-nitro-N-nitroguanidine (NTG), UVlight, or to a spontaneously occurring mutant. A mutant may have beensubjected to several mutagenization treatments (a single treatmentshould be understood one mutagenization step followed by ascreening/selection step), but it is presently preferred that no morethan 20, or no more than 10, or no more than 5, treatments (orscreening/selection steps) are carried out. In a presently preferredmutant, less than 1%, less than 0.1%, less than 0.01%, less than 0.001%or even less than 0.0001% of the nucleotides in the bacterial genomehave been mutated, i.e. substituted, inserted or deleted, compared tothe mother strain. Preferably, the genome of the mutant has at least 99%sequence identity to the genome of the mother strain and said mutantexhibits AhR agonist activity.

In an embodiment, the composition of the invention comprises aninactivated Coprococcus bacterium, i.e. at least one inactivatedCoprococcus strain. Preferably, said strain is a Coprococcus comesbacterium. More preferably, said strain is DSM 33359 or a mutantthereof. In this embodiment, it is not excluded that the compositionalso comprises other bacterial or culture components such as livingbacteria and/or cell debris and/or culture medium. Inactivated bacteriaare not able to grow when cultured in a suitable medium. Preferably,inactivated bacteria are killed bacteria. Bacteria can be inactivated byany means known by the skilled person such as heat treatment.Inactivated bacteria can be preserved before administration by anymethod known by the skilled person such as lyophilization and subsequentstorage, preferably at temperatures ranging from +4° C. to −80° C.

In a preferred embodiment, the composition of the invention comprises aliving Coprococcus bacterium, i.e. at least one living Coprococcusstrain. Preferably, said strain is a Coprococcus comes bacterium. Morepreferably, said strain is DSM 33359 or a mutant thereof. In thisembodiment, it is not excluded that the composition also comprises otherbacterial or culture components such as dead bacteria and/or cell debrisand/or culture medium. Living bacteria may be preserved beforeadministration by freezing with liquid nitrogen, gradual freezing orlyophilization and subsequent storage, preferably at temperaturesranging from +4° C. to −80° C.

In another embodiment, the composition of the invention comprises aculture extract of a Coprococcus bacterium, preferably a culture extractof a Coprococcus comes bacterium, more preferably a culture extract ofDSM 33359 strain or a mutant thereof.

As used herein, the term “culture extract” refers an extract obtainedfrom the culture of a Coprococcus bacterium, preferably a Coprococcuscomes bacterium, more preferably DSM 33359 strain or a mutant thereof,in a suitable cell culture medium and under suitable conditions, saidextract exhibiting AhR agonist activity. The AhR agonist activity of theextract may be assessed as described above. The extract may be anyfraction obtained from the culture or from bacterial cells, such as aculture supernatant, cell debris, cell walls, DNA or RNA extracts,protein extracts, and in general any preparation derived from bacterialcells or cell culture by chemical, physical and/or enzymatic treatments.The culture extract may be free of intact bacterial cells or may containsome residual intact bacterial cells, preferably less than 10³ cells permL.

In preferred embodiments, the culture extract is a culture supernatant.As used herein, the term “culture supernatant” refers a supernatantobtained from growing a Coprococcus bacterium, preferably a Coprococcuscomes bacterium, more preferably DSM 33359 strain or a mutant thereof,in a suitable cell culture medium and under suitable conditions, saidculture supernatant exhibiting AhR agonist activity. In particular, thisterm refers to the liquid broth remaining when cells grown in a mediumare separated from the culture medium by for example centrifugation,filtration, sedimentation, or other means well known in the art.Optionally, the culture supernatant may be further diluted,concentrated, dried and/or lyophilized. The culture supernatant may befree of living bacterial cells or may contain some residual livingbacterial cells, preferably less than 10³ cells per mL. The culturesupernatant may also comprise dead bacteria and/or cell components suchas cell debris.

In a particular embodiment, the culture supernatant is obtained bygrowing Coprococcus bacterium, preferably Coprococcus comes bacterium,more preferably strain DSM 33359 or a mutant thereof, in a suitablemedium, separating bacterial cells from the culture medium, e.g. bycentrifugation, filtration or sedimentation, and recovering the culturesupernatant.

In another particular embodiment, the culture supernatant is obtained bygrowing Coprococcus bacterium, preferably Coprococcus comes bacterium,more preferably strain DSM 33359 or a mutant thereof, in a suitablemedium, lysing bacterial cells in the culture medium, and recovering theculture supernatant (comprising dead bacteria and/or cell debris, i.e.the lysate).

In a further particular embodiment, the culture supernatant is obtainedby growing Coprococcus bacterium, preferably Coprococcus comesbacterium, more preferably strain DSM 33359 or a mutant thereof, in asuitable medium, lysing bacterial cells in the culture medium,separating bacterial cells and cell debris from the culture medium, e.g.by centrifugation, filtration or sedimentation, and recovering theculture supernatant.

Optionally, in any of these embodiments, the culture supernatant may befurther diluted or concentrated. In some preferred embodiments, theculture supernatant is dried or lyophilized.

In a further embodiment, the composition comprises a Coprococcusbacterium, preferably Coprococcus comes bacterium, more preferablystrain DSM 33359 or a mutant thereof, and a culture extract thereof,preferably a culture supernatant thereof. Preferably, the compositioncomprises a living Coprococcus bacterium. In said embodiment, thecomposition may comprise a cell culture comprising a Coprococcusbacterium. As used herein, the term “cell culture” refers to the mix ofbacterial cells and liquid broth used to culture these cells. Inparticular, the cell culture may be obtained by inoculating a suitableliquid broth with a living Coprococcus bacterium, incubating said mix insuitable conditions (in particular in anaerobic conditions), andrecovering cell culture. Alternatively, the composition may comprise aCoprococcus bacterium, preferably a living Coprococcus bacterium, and aculture extract thereof, preferably a culture supernatant thereof,obtained by any method disclosed above, in particular obtained bygrowing Coprococcus bacterium in a suitable medium, and a) separatingbacterial cells from the culture medium, e.g. by centrifugation,filtration or sedimentation, and recovering the culture supernatant; orb) lysing bacterial cells in the culture medium, and recovering theculture supernatant (comprising dead bacteria and/or cell debris, i.e.the lysate); or c) lysing bacterial cells in the culture medium,separating bacterial cells and cell debris from the culture medium, e.g.by centrifugation, filtration or sedimentation, and recovering theculture supernatant. Optionally, the culture supernatant may be furtherdiluted, concentrated, dried and/or lyophilized.

In another embodiment, the composition comprises a Coprococcusbacterium, preferably a living Coprococcus bacterium, and a cultureextract, preferably a culture supernatant, of another Coprococcusbacterium. Preferably, the Coprococcus bacterium and/or the Coprococcusbacterium used to obtained the culture extract is a Coprococcus comesbacterium, more preferably is DSM 33359 strain or a mutant thereof.

In some particular embodiments, the composition does not comprise anyCoprococcus catus bacterium or any culture extract thereof, and/or anyCoprococcus eutactus bacterium or any culture extract thereof.

The composition may further comprise at least one additional activeingredient, in particular one or several additional bacterial probioticsand/or one or several AhR agonists.

The composition of the invention may comprise one or several additionalbacterial probiotics. The term “bacterial probiotic” has its generalmeaning in the art and refers to a useful bacterium that can bring abeneficial action to the host health, i.e. which is applicable to theprevention, treatment or cure of a disease or condition of the host,preferably a human being. This term may refer to a dead or livingbacterium. Preferably, this term refers to a living bacterium.

Preferably, one or several of these additional bacterial probioticsexhibit AhR activation properties. The AhR may be activated by thebacterial probiotic as AhR ligand, by a secretory substance produced bysaid bacterial probiotic and having AhR-activating potency, or by a deadmicrobial body or homogenate of said bacterial probiotic.

Preferably, said one or several bacterial probiotics are capable ofproducing an AhR agonist. Such bacterial probiotics can be selected, forexample, from the group consisting of bacteria belonging to the generaAllobaculum (e.g. Allobaculum stercoricanis), Adlercreutzia,Anaerostipes (e.g. Anaerostipes hadrus, Anaerostipes caccae andAnaerostipes butyraticus), Bifidobacterium, Propionibacterium,Bacteroides, Eubacterium, Enterococcus, Ruminococcus (e.g. Ruminococcusgnavus) and Faecalibacterium (e.g. Faecalibacterium prausnitzii),Escherichia coli, and lactic acid bacteria such as bacteria belonging tothe genera Lactobacillus (e.g. Lactobacillus reuteri, Lactobacillustaiwanensis, Lactobacillus johnsonii, Lactobacillus animalis,Lactobacillus murinus, Lactobacillus salivarius, Lactobacillus gasseri,Lactobacillus bulgaricus, and Lactobacillus delbrueckii subsp.Bulgaricus) and Streptococcus (e.g. Streptococcus thermophilus). In aparticular embodiment, the composition may comprise one or severalbacterial probiotics selected from the group consisting of bacteriabelonging to the genera Allobaculum, Adlercreutzia, Propionibacterium,Enterococcus, Escherichia coli, Lactobacillus and Streptococcus.

In preferred embodiments, said one or several additional probiotics areselected from the group consisting of strains CNCM I-5019 (Lactobacillustaiwanensis), CNCM I-5020 (Lactobacillus murinus), CNCM I-5021(Lactobacillus animalis), CNCM I-5022 (Lactobacillus reuteri) and CNCMI-5023 (Lactobacillus reuteri), deposited on Sep. 30, 2015 at theCollection Nationale de Cultures de Microorganismes (CNCM, InstitutPasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France),Ruminococcus gnavus ATCC 29149, Lactobacillus salivarius DSM 20555,Lactobacillus reuteri DSM 20016, Lactobacillus gasseri DSM 20243,Faecalibacterium prausnitzii A2-165, Escherichia coli MG1665,Anaerostipes hadrus DSM 3319, Anaerostipes caccae DSM 14662,Anaerostipes butyraticus DSM 22094 and Allobaculum stercoricanis DSM13633.

The composition of the invention may also comprises one or several AhRagonists.

The AhR agonists are small organic molecules or peptides and includesynthetic and naturally occurring compounds. The term “AhR agonist” hasits general meaning in the art and refers to a compound that activatesthe AhR, preferably that selectively activates the AhR. AhR agonists maybe a natural AhR ligand or any compound that can directly or indirectlystimulate the signal transduction cascade related to the AhR. As usedherein, the term “selectively activates” refers to a compound thatpreferentially binds to and activates AhR with a greater affinity andpotency, respectively, than its interaction with the other members ofbHLH-PAS transcription factors family. Compounds that prefer AhR, butthat may also activate other sub-types, as partial or full agonists arealso contemplated. Tests and assays for determining whether a compoundis an AhR agonist are well known by the person skilled in the art suchas described in Ji et al., 2015, Dig Dis Sci, 60, 1958-1966; Furumatsuet al., 2011, Dig Dis Sci, 56, 2532-2544; WO 2013/171696; WO2012/015914; U.S. Pat. No. 6,432,692. In vitro and in vivo assays may beused to assess the potency and selectivity of the candidate agents toinduce AhR activity.

Examples of AhR agonists include, but are not limited to, halogenatedaromatic hydrocarbons (e.g. polychlorinated dibenzodioxins,dibenzofurans and biphenyls), polycyclic aromatic hydrocarbons (e.g.3-methylcholanthrene, benzo-a-pyrene, benzanthracenes andbenzoflavones), indoles derivatives, kynurenine, kynurenic acid,tryptophan catabolites of the microbiota such as indole-3-aldehyde(IAid), indole propionic acid, tryptamine, indole 3-acetate, 3-indoxylsulfate, 6-formylindolo(3,2-b)carbazole (FICZ), indolo(3,2-b)carbazole(ICZ), 2-(1′H-1-indole-3′-carbonyl)-thiazole-4-carboxylic acid methylester (ITE) or its precursor2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylate (ITC), and analogsthereof disclosed in U.S. Pat. No. 7,419,992,2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), polycyclic aromatichydrocarbon (PAH), polychlorinated biphenyl (PCB), 3-indoxyl-sulfate(135),1-(4-Methylphenyl)-2-(4,5,6,7-tetrahydro-2-imino-3(2H)-benzothiazolyl)ethanonehydrobromide (Pifithrin-α hydrobromide),(2′Z,3′E)-6-Bromo-1-methylindirubin-3T-oxime (MeB10), tryptophanderivatives such as indigo dye and indirubin, flavonoids, biphenyls,tetrapyrroles such as bilirubin, the arachidonic acid metaboliteslipoxin-A4 and prostaglandin G, modified low-density lipoprotein andseveral dietary carotenoids (Denison et al., 2002, Chem. Biol. Interact.141, 3-24; Denison et al., 2003, Annu. Rev. Pharmacol. Toxicol. 43,309-334; Adachi J et al., 2001, J. Biol. Chem., 276, 31475-1478; Sinal CJ and Bend J R, 1997, Mol. Pharmacol., 52, 590-9; Seidel S D, et al.,2001, J. Biochem. Mol. Toxicol., 15, 187-196; McMillan B J and BradfieldC A, 2007, Proc. Natl. Acad. Sci. U.S.A., 104, 1412-1417; Stevens etal., 2009, Immunology., 127, 299-311), AhR agonists disclosed in Bissonet al., 2009, J. Med. Chem, 52, 5635-5641 (e.g.5-hydroxy-7-methoxyflavone, 7-methoxyisoflavone, 6-methylflavone,3-hydroxy methylflavone, pinocembrin (5,7-dihydroxyflavanone) and7,8,2′-trihydroxyflavone), compound VAF347[4-(3-chlorophenyl)-N-[4-(trifluoromethyl)phenyl]pyrimidin-2-amine], andits pro-drug version VAG539[4-(3-chloro-phenyl)-pyrimidin-2-yl]-(4-trifluoromethyl-phenyl)-carbamicacid 2-[(2-hydroxy-ethyl)-methyl-amino]-ethyl ester] (Lawrence B P,2008, Blood, 112, 1158-1165), semaxanib (5U5416)[3-(3,5-dimethyl-1H-pyrrol-2-ylmethylene)-1,3-dihydro-indole-2-one](Mezrich J D, et al. (2012) PLoS ONE 7(9): e44547), selective AhRmodulators (SAhRM) (e.g. diindolylmethane (DIM), methyl-substituteddiindolylmethanes, dihalo- and dialkylDIM analogs, β-naphthoflavone(βNF) (5,6 benzoflavone (5,6 BZF) and moieties described, for example,in Safe et al., 2013, Toxicol Sci., 135, 1-16; Furumatsu et al., 2011,Dig Dis Sci, 56, 2532-2544; and WO 2012/015914), compounds described inWO 2012/015914 (e.g. CB7950998), 1,4-dihydroxy-2-naphthoic acid (DHNA)and natural AhR Agonists (NAhRAs) disclosed in WO 2013/171696 and WO2009/093207, Mexiletine, Nimidipine, Flutamide, Atorvastatin,Leflunomide, and Ginseng (Hu W et al, 2007, Mol Pharmacol., 71, 1475-86,O'Donnell E F, et al, 2010, PLoS One, 5(10). pii: e13128; Wang Y, et al,2008, Eur J Pharmacol., 601, 73-78).

In a particular embodiment, said one or several AhR agonists areselected from the group consisting of approved drugs having an agonisteffect on AhR, preferably from the group consisting of Mexiletine,Nimidipine, Flutamide, Atorvastatin, Leflunomide and Ginseng.

As additional active ingredients, the composition may also comprise oneor several drugs useful in the treatment of metabolic syndrome andassociated disorders, or useful in the treatment of inflammatory boweldiseases. Examples of drugs useful in the treatment of inflammatorybowel diseases include, but are not limited to, corticosteroids,5-aminosalicylates, immunosuppressors such as cyclosporin, azathioprine,6-mercaptopurine, methotrexate, anti-TNF agents (such as infliximab,adalimumab, golimumab, certolizumab), anti-integrin agents (such asnatalizumab, vedolizumab) anti-IL12 and/or -IL23 antibodies (such asustekinumab), JAK inhibitors (such as tofacitinib), antibiotics,anti-diarrheals, pain relievers, iron supplements, vitamin B-12, calciumand vitamin D.

In a particular embodiment, the composition consists essentially in oneor several Coprococcus bacteria, preferably one or several livingCoprococcus bacteria, and/or culture extract thereof, preferably culturesupernatant thereof. As used herein, the term “consists essentially in”is intended to refer to a composition that does not comprise any otheractive ingredient, in particular does not comprise any other bacterium,especially any other bacterial probiotic.

In preferred embodiments, the composition of the invention is apharmaceutical composition comprising a Coprococcus bacterium as definedabove, preferably a living Coprococcus bacterium, and/or a cultureextract thereof, preferably a culture supernatant thereof, and apharmaceutically acceptable excipient.

As used herein, the term “pharmaceutically acceptable” refer tomolecular entities and compositions that do not produce an adverse,allergic or other untoward reaction when administered to a mammal,especially a human, as appropriate. A pharmaceutically acceptableexcipient refers to a non-toxic solid, semi-solid or liquid filler,diluent, encapsulating material or formulation auxiliary of any type.The pharmaceutically acceptable excipients that can be used in thecomposition according to the invention are well known to the skilledperson and may vary according to the disease to be treated and theadministration route.

The composition of the invention can be administered by any methodsuitable for depositing in the gastrointestinal tract, preferably thesmall intestine and/or the colon, of the subject to be treated. Inparticular, the composition can be administered by enteral or parenteralroute, preferably by oral, sublingual, subcutaneous, intramuscular,intravenous, transdermal, local or rectal administration route.Preferably, the composition of the invention is administered, or isadapted to be administered, by rectal or oral route.

In an embodiment, the pharmaceutical composition is to be administeredby oral route. For oral administration, the pharmaceutical compositioncan be formulated into conventional oral dosage forms such as tablets,capsules, powders, granules and liquid preparations such as syrups,elixirs, and concentrated drops. Nontoxic solid carriers or diluents maybe used which include, for example, pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, talcum,cellulose, glucose, sucrose, magnesium, carbonate, and the like. Forcompressed tablets, binders, which are agents which impart cohesivequalities to powdered materials, are also necessary. For example,starch, gelatin, sugars such as lactose or dextrose, and natural orsynthetic gums can be used as binders. Disintegrants may also benecessary in the tablets to facilitate break-up of the tablet.Disintegrants include starches, clays, celluloses, algins, gums andcrosslinked polymers. Moreover, lubricants and glidants may also beincluded in the tablets to prevent adhesion to the tablet material tosurfaces in the manufacturing process and to improve the flowcharacteristics of the powder material during manufacture. Colloidalsilicon dioxide is most commonly used as a glidant and compounds such astalc or stearic acids are most commonly used as lubricants. Well-knownthickening agents may also be added to compositions such as corn starch,agar, natural or synthetic gums, resins, methylcellulose, sodiumcarboxymethylcellulose, guar, xanthan and the like. Preservatives mayalso be included in the composition, including methylparaben,propylparaben, benzyl alcohol and ethylene diamine tetraacetate salts.

In some particular embodiments, the composition may be a beverage ordrink composition, a food composition or a feedstuff composition.

Preferably, for oral administration, the composition is in agastro-resistant oral form allowing the active compounds contained inthe composition, to pass the stomach and be released into the intestine.The material that can be used in enteric coatings includes, for example,alginic acid, cellulose acetate phthalate, plastics, waxes, shellac andfatty acids (e.g. stearic acid or palmitic acid).

In another embodiment, the pharmaceutical composition is to beadministrated by rectal route. Suitable rectal-route forms include, butare not limited to, suppository and enema. In particular, the activecompounds can be incorporated into any of the known suppository bases bymethods known in the art. Examples of such bases include cocoa butter,polyethylene glycols (carbowaxes), polyethylene sorbitan monostearate,and mixtures of these with other compatible materials to modify themelting point or dissolution rate.

Compositions according to the invention may be formulated to release theactive ingredients substantially immediately upon administration or atany predetermined time or time period after administration.

The composition of the invention is used in the treatment of a diseaseselected from the group consisting of metabolic syndrome and associateddisorders, and inflammatory bowel diseases.

As used herein, the term “treatment”, “treat” or “treating” refers toany act intended to ameliorate the health status of patients such astherapy, prevention, prophylaxis and retardation of the disease. Incertain embodiments, such term refers to the amelioration or eradicationof the disease or symptoms associated with it. In other embodiments,this term refers to minimizing the spread or worsening of the diseaseresulting from the administration of one or more therapeutic agents,e.g. the composition of the invention, to a subject with such a disease.

In some embodiments, the disease is the metabolic syndrome or anyassociated disorder. The metabolic syndrome is defined by a clusteringof at least three of the five following medical conditions:

-   -   Abdominal (central) obesity;    -   Elevated blood pressure;    -   Elevating fasting plasma glucose;    -   High serum triglycerides; and    -   Low high-density lipoprotein (HDL) levels.

It is important to note that obesity doesn't equate to metabolicsyndrome. Patients who are of normal weight may also have a metabolicsyndrome and, on the opposite, obese people may not have a metabolicsyndrome. Indeed, as mentioned above, the metabolic syndrome isestablished if the obesity is an abdominal obesity and if at least twoother medical conditions are observed.

According to the International Diabetes Federation, a consensusworldwide definition of the metabolic syndrome (2006) is: Centralobesity (defined as waist circumference with ethnicity-specific values)and any two of the following:

-   -   Elevated blood pressure (BP): systolic BP>130 or diastolic BP>85        mm Hg, or treatment of previously diagnosed hypertension;    -   Elevating fasting plasma glucose (FPG): >100 mg/dL (5.6 mmol/L),        or previously diagnosed type 2 diabetes;    -   High serum triglycerides refers to >150 mg/dL (1.7 mmol/L) or        specific treatment for this lipid abnormality.    -   Low high-density lipoprotein (HDL) levels: <40 mg/dL (1.03        mmol/L) in males, <50 mg/dL (1.29 mmol/L) in females, or        specific treatment for this lipid abnormality.

If BMI is >30 kg/m², central obesity can be assumed and waistcircumference does not need to be measured.

Metabolic syndrome is associated with the risk of developing otherdisorders, i.e. associated disorders. Preferably, these associateddisorders are selected from the group consisting of cardiovasculardiseases, in particular coronary heart disease, especially heart attackand stroke, insulin resistance, glucose intolerance, type 2 diabetes,non alcoholic fatty liver disease, especially non-alcoholicsteatohepatitis, and lipodystrophy. In a particular embodiment, theseassociated disorders are selected from the group consisting ofcardiovascular diseases, in particular coronary heart disease,especially heart attack and stroke, insulin resistance, glucoseintolerance, non alcoholic fatty liver disease, especially non-alcoholicsteatohepatitis, and lipodystrophy.

In some other embodiments, the disease is an inflammatory bowel disease.As used herein, “inflammatory bowel diseases” or “IBD” refers to any ofa variety of diseases characterized by inflammation of all or part ofthe intestines. Examples of inflammatory bowel diseases include, but arenot limited to, Crohn's disease (such as enteritis, ileitis, colitis,ileocolitis, gastroduodenal and perianal Crohn's), ulcerative colitis(such as ulcerative enterocolitis, ulcerative ileocolitis, ulcerativeproctitis, ulcerative rectosigmoiditis, pseudopolyposis of colon,mucosal proctocolitis, and left-sided or extensive ulcerative colitis),indeterminate colitis (IC), other noninfective gastroenteritis,enteritis, enterocolitis and colitis (such as non-microscopic colitisincluding collagenous colitis and lymphocytic colitis,radiation-induced, toxic-induced, allergic-induced, dietetic-induced,ischemic, eosinophilic gastroenteritis, enteritis, enterocolitis orcolitis, segmental colitis associated with diverticula, diversioncolitis and Behcet's colitis) as well as pouchitis. In some particularembodiments, the inflammatory bowel disease is not antibiotic associatedcolitis or more particularly is not Clostridium difficile associateddiarrhea.

Preferably, the inflammatory bowel disease is selected from the groupconsisting of Crohn's disease and ulcerative colitis, more preferably isulcerative colitis.

The subject to be treated with the composition of the invention is ananimal, preferably a mammal, even more preferably a human, includingadult, child, newborns and human at the prenatal stage. As used herein,the terms “subject”, “individual” and “patient” are interchangeable.

In an embodiment, the subject exhibits a decreased AhR activity,especially in a feces sample, more particularly a decreased AhR agonistactivity of the gut microbiota. In a particular embodiment, the activityof AhR is measured for the subject. Preferably, the AhR activity is theactivity of the microbiota and is measured in a feces sample.

In a preferred embodiment, the subject exhibits a decreased AhR activityby comparison with a predetermined reference value. As used herein, the“reference value” refers to a threshold value or a cut-off value. Thereference value can be determined experimentally, empirically, ortheoretically. Preferably, the reference value is derived from the AhRactivation level determined in one or several feces samples derived fromone or more healthy subjects, especially who are not suffering of ametabolic syndrome, an associated disorder and/or IBD. Preferably, thesubject having a decreased AhR activity exhibits a AhR activity which isat least 10% lower than the predetermined reference value, morepreferably at least 20%, 30%, or 50% lower than the predeterminedreference value.

The dosage of Coprococcus bacterial cells and/or culture extract may beappropriately adjusted according criteria such as age, symptoms, bodyweight, and intended application such as to obtain a therapeuticallyefficient amount. The term “therapeutically efficient amount” asemployed herein refers to the amount necessary for having a beneficialimpact on the disease to be treated, i.e. to prevent, remove or reduceat least one deleterious effect of the disease.

In embodiments wherein the disease to be treated is metabolic syndromeor an associated disorder, an therapeutically efficient amount ispreferably defined as the amount necessary for having an impact (i.e.i.e. to prevent, remove or reduce) on one of the five medical conditionsdefining the metabolic syndrome:

-   -   Abdominal (central) obesity (TOF1);    -   Elevated blood pressure;    -   Elevating fasting plasma glucose;    -   High serum triglycerides; and    -   Low high-density lipoprotein (HDL) levels.

In addition or alternatively, the therapeutically efficient amount maybe defined as the amount necessary for having an impact on the insulinsensitivity, the glucose tolerance, the weight gain and/or theintestinal inflammation due to HFD. In a preferred embodiment, thetherapeutically efficient amount has an impact on several of theseconditions.

In embodiments wherein the disease to be treated is IBD, antherapeutically efficient amount is preferably defined as the amountnecessary for having an impact on intestinal inflammation or any symptomof the disease such as diarrhea, fever or pain.

In some embodiments, the composition of the invention may comprise from10³ to 10¹¹ living Coprococcus bacterial cells, preferably Coprococcuscomes bacterial cells, more preferably bacterial cells of DSM 33359 or amutant thereof, per mg of composition and/or from 0.001 mg to 1000 mg ofdried or lyophilized culture supernatant per g of composition.

In particular, the amount of Coprococcus bacteria, preferablyCoprococcus comes bacteria, more preferably DSM 33359 or a mutantthereof, ingested per day may be from 0.01×10¹¹ to 100×10¹¹ cells/body,preferably 0.1×10¹¹ to 10×10¹¹ cells/body, and more preferably 0.3×10¹¹to 5×10¹¹ cells/body.

The content of Coprococcus bacteria, preferably Coprococcus comesbacteria, more preferably DSM 33359 or a mutant thereof, contained in anorally ingested composition of the invention may be for example from 1%to 100% (w/w, i.e. bacteria dry weight/total dry weight of thecomposition), preferably from 1% to 75% (w/w), and more preferably from5% to 50% (w/w).

The composition of the invention may be administered as a single dose orin multiple doses.

In some embodiments, the composition is to be administered regularly,preferably between every day and every month, more preferably betweenevery day and every two weeks, more preferably between every day andevery week. In some particular embodiments, the composition is to beadministered every day.

The duration of treatment with the composition of the invention may becomprised between 1 day and several years, preferably between 1 day andone year, more preferably between 1 day and 6 months.

The present invention also relates to a method of (i) selecting asubject suffering of a disease selected from metabolic syndrome andassociated disorders, and inflammatory bowel diseases, for a treatmentwith a composition of the invention or (ii) determining whether asubject affected with a disease selected from metabolic syndrome andassociated disorders, and inflammatory bowel diseases, is susceptible tobenefit from a therapy with a composition of the invention, wherein adecreased AhR activity in the subject, especially in a feces sample fromthe subject, indicates that a therapy with the composition of theinvention is suitable. More particularly, the method comprises the stepsof: i) determining the AhR agonist activity of the microbiota in a fecessample obtained from the subject, ii) comparing the level determined atstep i) with a predetermined reference value, and iii) selecting thesubject as suitable for the treatment when the level determined at stepi) is lower than the predetermined reference value. The predeterminedreference value is preferably a reference value derived from the AhRactivation level determined in one or several feces samples derived fromone or more healthy subjects, especially who are not suffering of ametabolic syndrome, an associated disorder and/or IBD.

The AhR activation level of the microbiota in a feces sample obtainedfrom the subject may be assessed by any method described above.Additionally or alternatively, the AhR activation level of themicrobiota in a feces sample obtained from the subject may be assessedby measuring tryptophan metabolism, i.e. by measuring Tryptophan (Trp),kynurenine (Kyn) and indole-3-acetic acid (IAA) concentrations (or othertryptophan metabolites), and optionally calculating Kyn/Trp, IAA/Trp andKyn/IAA concentration ratios. Additionally or alternatively, the AhRactivation level may also be assessed using colon samples obtained fromthe subject by analyzing the expression of AhR target genes (such asinterleukins IL-22 and IL-17), measuring IL-17⁺ and IL-22⁺ cells number,measuring AhR and chaperone proteins heterodimerization, measuring AhRnuclear translocation, or measuring AhR binding to its dimerizationpartner (AhR nuclear translocator (ARNT)).

Preferably, the subject is selected for the treatment with thecomposition of the invention if the level determined at step i) is atleast 10% lower than the predetermined reference value, more preferablyat least 20%, 30%, or 50% lower than the predetermined reference value.

In another aspect, the present invention also relates to Coprococcuscomes strain deposited with the Deutsche Sammlung von Mikroorganismenund Zellkulturen (DSMZ) under accession number DSM 33359, or a mutantthereof. The present invention also relates to Coprococcus comes straindeposited with the Deutsche Sammlung von Mikroorganismen undZellkulturen (DSMZ) under accession number DSM 33359, or a mutantthereof, for use in the treatment of a disease selected from the groupconsisting of metabolic syndrome and associated disorders, andinflammatory bowel diseases. It also relates to the use of Coprococcuscomes strain deposited with the Deutsche Sammlung von Mikroorganismenund Zellkulturen (DSMZ) under accession number DSM 33359, or a mutantthereof, for the manufacture of a medicament for the treatment of adisease selected from the group consisting of metabolic syndrome andassociated disorders, and inflammatory bowel diseases. It furtherrelates to a method for treating a disease selected from the groupconsisting of metabolic syndrome and associated disorders, andinflammatory bowel diseases, in a subject, said method comprisingadministering to the subject Coprococcus comes strain deposited with theDeutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) underaccession number DSM 33359, or a mutant thereof.

All the references cited in this description are incorporated byreference in the present application. Others features and advantages ofthe invention will become clearer in the following examples which aregiven for purposes of illustration and not by way of limitation.

EXAMPLES

Methods

Mice

Male and female C57BL/6JRj mice were purchased from Janvier (France).AhR^(−/−) mice on the C57BL/6JRj background were obtained from theJackson laboratory (JAX stock #002831) (Fernandez-Salguero et al.Science. 1995, 268, 722-726) and were bred at Saint-Antoine ResearchCenter. Il22^(−/−) mice (Kreymborg et al., J. Immunol. 2007, 179,8098-8104) were obtained and bred at the Transgenose Institute(TAMM-CNRS, Orleans, France). All the mice were housed at the IERPfacility (INRA, Jouy-en-Josas, France), which is accredited by theFrench “Direction Départementale de la Protection des Populations(DDPP78)”. All experiments were performed in accordance with the Comited'Ethique en Experimentation Animale (COMETHEA C2EA-45, Jouy en Josas,France).

Bacterial Strain and Growth Conditions

Coprococcus comes was grown at 37° C. in LYHBHI medium (Brain—heartinfusion medium supplemented with 0.5% yeast extract (Difco) and 1%hemine (Sigma-Aldrich)) supplemented with cellobiose (1 mg/ml;Sigma—Aldrich), maltose (1 mg/ml; Sigma-Aldrich), cysteine (0.5 mg/ml;Sigma-Aldrich), vitamin K1 (0.0002%; Sigma-Aldrich) and vitamin K3(0.0002%; Sigma-Aldrich) in an anaerobic chamber (0 ppm O₂; 2% H₂).Different conditions of bacteria were used in both models, DSS treatmentand metabolic syndrome: bacteria only, bacteria supplemented with thesupernatant, supernatant or killed bacteria. After 24 h of incubation,Coprococcus comes culture was centrifuged at 6,000 g during 10 min at 4°C. Supernatant was harvested and supplemented with 16% of glycerol forbetter preservation at −80° C. The pellets were resuspended in completeLYHBHI medium supplemented with 16% of glycerol or with supernatant alsosupplemented with 16% of glycerol. Killed bacteria were obtained afterboiling at 100° C. during 30 min, then centrifugation (6,000 g, 10 min,4° C.) was performed and the pellet was resuspended in complete LYHBHImedium supplemented with 16% of glycerol. Mice were inoculated dailywith 10⁹ CFU of Coprococcus comes or vehicle (complete LYHBHI mediumsupplemented with 16% of glycerol) throughout the protocols.

AhR/Luciferase Reporter Assay

The H1L1.1c2 cell line was used as described before (Lamas et al., 2016,Nat Med. June; 22(6):598-605). Control (2, 10 or 20% LYHBHI),supernatant of 24h bacteria culture were used and incubated with theH1L1.1c2 cell line during 24h. AhR activity was calculated bysubtracting the luminescence of the control (2, 10 or 20% LYHBHI) fromthe luminescence obtained with the samples multiplied by thecytotoxicity value (LDH dosage).

DSS Model

C57BL/6JRj from Janvier lab, 7 weeks old, were housed in our specificpathogen free animal facility at IERP (INRA, Jouy en Josas, France).Five to ten female mice per group were used in all experiment, weremaintained in a temperature controlled (23° C.) facility with a strict12 h light/dark cycles and were given free access to food and water.After one acclimation week, we changed water with DSS 2% (MP Biomedical)during 7 days (day 0 to day 7), and a recovery period was practiceduntil day 12 (during 5 days). Body weight loss and DAI (disease activityindex) were performed daily. After 12 days the samples were harvested.

Body weight loss 0 No loss 1 1-5% loss of body weight 2 6-10% loss ofbody weight 3 10-20% loss of body weight 4 >20% loss of body weightStool consitency 0 Normal Feces 1 Loose stool 2 Watery diarrhea 3 Slimydiarrhea, little blood 4 Severe watery diarrhea with blood Blood instool 0 No blood 1 Presence of blood 2 Visible blood-low quantity 3Visible blood-medium quantity 4 Visible blood-high quantity

Colon Histology

Colon distal part (cut in three parts) was harvested at day 12 after DSSmodel and put directly in PFA (Rothi®—Histofix 4%) during 48h andtransferred in ethanol 70% until the standard pre-fixation automat andparaffin embedded. HES coloration was practiced on 5 μm cut. Scoring wasperformed on the worst cut and scored as previously described (Sokol etal., Gastroenterology. 2013 September; 145(3):591-601.e3.).

MLN Stimulation

MLN (mesenteric lymph node) cells was harvested and grinded on 40 μMcells strainer. Cells were resuspended in complete RPMI (10% SVF,Pen/Strep) and counted with Accuri cytometer (BD). Two millions of cellswere put on 24 wells plate and stimulated with PMA (50 ng/ml, Sigma) andionomycin (750 ng/ml, Sigma) during 48 h at 37° C. Supernatants ofstimulation were recovered and ELISA (Mabtech) against IL-17A, IL-10,IFN-γ and IL-22 were performed according to the manufacturer'sinstructions.

Real Time PCR

Samples were extracted with Rneasy Mini kit (Qiagen). Total RNA (1 μg)was reverse transcribed using LunaScript™ Reverse Transcription SuperMixKit (Biolabs). The mRNA levels for the genes of interest were examinedby quantitative RT-PCR using the Luna® Universal qPCR Master Mix(Biolabs) or Universal Probe qPCR Master Mix (Biolabs) according to themanufacturer's protocol. Primers and probes used were obtained fromQiagen or Thermofisher: Cyp1a1 (TaqMan, Thermofisher, Mm00487218_m1),IL-22 (TaqMan, Thermofisher, Mm01226722_g1), Reg3γ(TTCCTGTCCTCCATGATCAAAA (SEQ ID NO: 1)/CATCCACCTCTGTTGGGTTCA (SEQ ID NO:2)), Reg3β (ATGCTGCTCTCCTGCCTGATG (SEQ ID NO:3)/CTAATGCGTGCGGAGGGTATATTC (SEQ ID NO: 4)), AhRR (GGAGTCTCTCAATGGCTTCG(SEQ ID NO: 5)/CCGAGTACTCTGAGGGCAAG (SEQ ID NO: 6)). Relative levels ofmRNA expression were normalized to HPRT1 (QT00166768) mRNA levels usinga comparative method (2-ΔΔCt). Non-reverse-transcribed RNA samples andwater were included as negative controls.

Metabolic Syndrome

C57BL/6JRj from Janvier lab, 5 weeks old, were housed in our specificpathogen free animal facility at IERP (INRA, Jouy en Josas, France). Sixto eight male mice per group were used in all experiment, weremaintained in a temperature controlled (23° C.) facility with a strict12h light/dark cycles and were given free access to food and water. MaleC57BL/6JRj mice at 5 weeks of age were fed ad libitum with a purifiedcontrol diet (Cony, Envigo Md.120508) or high fat diet (38% kcal fat,dominantly milk fat, Envigo Md.97222) for 12 weeks. Animals were weighedweekly, and weekly food consumption was measured in each cage. Allanimals were fasted during the night before the sacrifice and theneuthanized by cervical dislocation, and appropriate tissues wereharvested.

Oral Glucose Tolerance Test

OGGT was performed 3-7 days before sacrifice. Mice were fasted byremoval of the food and bedding during the night before the experiment.After 15-16 hours of fasting, a glucose solution (2 g/kg per mice) wasadministered by oral gavage. Blood glucose levels at time 0 (fastingglucose, taken before glucose gavage) and 15, 30, 60 and 120 min afterglucose gavage were analyzed using a OneTouch glucometer (Roche). Theglucose level was plotted relative to time, and the AUC was calculatedaccording to the trapezoidal rule.

Intraperitoneal Insulin Tolerance Test

ITT was performed 3-7 days before sacrifice. Mice were fasted by removalof the food and bedding 4 hours before the beginning of the experiment.After 4 hours of fasting, an insulin solution (0.5 U/kg) wasadministered intraperitoneally. Blood glucose levels at time 0 (fastingglucose, taken before glucose gavage) and at 15, 30, 60 and 120 minafter the insulin challenge were analyzed using a OneTouch glucometer(Roche). The glucose levels were plotted relative to time, and the AUCwas calculated according to the trapezoidal rule.

Measurements of Plasma Parameters

Blood samples were collected in heparin-coated tubes via cardiacpuncture and centrifuged. The plasma samples were then stored at −80° C.until further analysis. Measurements of plasma cholesterol, high-densitylipoprotein (HDL) were performed by using the Biochemistry Platform(CRI, UMR 1149, Paris) with an Olympus AU400 Chemistry Analyzer.

Liver Histology and Hepatic Triglyceride Measurement

A slice of the left lobe of the liver was fixed in 4% PFA for 48 h andthen transferred to ethanol, fixed in paraffin, trimmed, processed,sectioned into slices that were approximately 5 μm thick, mounted on aglass slide and stained with HES. Hepatic lipids were evaluated andquantified blindly by using the ImageJ software as previously described(Crane et al., 2015, Nat Med 21, 166-172; Schneider et al., 2012, NatMethods 9, 671-675).

Statistical Analysis

Data were analyzed using Prism version 7 (Graphpad Software, San Diego,USA). The non-parametric Mann Withney test or the parametric one-wayANOVA tests with multiple Bonferroni's comparison tests were performed.Values are expressed as mean±SEM. Statistical significance was definedat a p-value ****<0.0001, ***<0.001, **<0.01, *<0.05.

Results

Coprococcus comes Supernatant is Associated with a High AhR Activity

The inventors assayed the capacity of several bacterial strains toproduce AhR agonists. Different supernatant of bacteria have been testedon H1L1.1c2, an AhR reporter cell line and they found that Coprococcuscomes supernatant is associated with a high AhR activity (data notshown).

C. comes Supernatant Protects from DSS-Induced Colitis

DSS-induced colitis mice were orally treated with C. comes(bacteria+supernatant, supernatant or killed bacteria). After 12 days ofDSS-induced colitis, body weight loss, disease activity index (DAI),colon length and histological score were assessed (FIG. 1 ). It wasfound that the administration of C. comes and/or its supernatant protectfrom DSS-induced colitis. In particular, C. comes supernatant was shownto induce a significant reduction of the production of IL-17, apro-inflammatory cytokine, after MLN cells stimulation.

C. comes Protection Against DSS-Induced Colitis is AhR Dependent.

The inventors demonstrated that C. comes supernatant is able to activateAhR in vitro in H1L1.1c2 cells (FIG. 2A). This result was confirmed invivo using WT mice after C. comes supernatant gavage during 3 weeks.Activation of AhR was assessed by measuring expression of Cyp1a P450cytochrome and AhR repressor (AhRR) in colon and liver by qPCR. Anincrease of these expression levels in colon of mice treated with C.comes supernatant indicates that the supernatant is able to activate AhRin vivo (FIG. 2B).

In order to confirm that the protection is due to AhR activation, bodyweight loss, DAI, colon length and histological score after DSS-inducedcolitis were measured in AhR−/− mice with or without C. comessupernatant gavage (FIGS. 2C-G). No difference was observed between thegroup treated with the negative control and the group treated with C.comes supernatant. The AhR-dependent mechanism of the C. comes was thusconfirmed by its lack of efficacy to protect AhR−/− mice fromDSS-induced colitis.

C. comes Protection Passes Though IL-22 Secretion

After 3 weeks of C. comes supernatant gavage, expression levels ofReg3γ, Reg3B and IL-22 were measured in colon of WT mice. It wasobserved that expression of IL-22 as well as Reg3γ and Reg3β, two genesinvolved in IL-22 pathway, was significantly increased by theadministration of C. comes supernatant (FIG. 3A).

In order to confirm that the protection involves Il-22 pathway, bodyweight loss, DAI, colon length and histological score were assessed inIL-22^(−/−) mice after 9 days of DSS-induced colitis (FIGS. 3B-F). Nodifference was observed between the group treated with the negativecontrol and the group treated with C. comes supernatant. This lack ofefficacy to protect IL22^(−/−) mice from DSS-induced colitis confirmsthat C. comes protection passes though IL-22 secretion.

Treatment with Coprococcus comes Alleviates Diet-Induced MetabolicImpairments

Diet-induced mice models of metabolic syndrome (HFD-fed mice) wereorally treated with C. comes (bacteria+supernatant, supernatant orkilled bacteria) during 12 weeks.

It was found that Coprococcus comes supplementation reduces body weightgain independently of food intake (FIGS. 4A-B). HFD-fed micesupplemented with Coprococcus comes also showed better glucose clearanceduring oral glucose tolerance test (OGGT) (FIGS. 4C-D) and insulinsensitivity during insulin tolerance test (ITT) (FIGS. 4E-F).Coprococcus comes supplementation also reduces features of hepaticsteatosis including lower hepatic lipids (FIG. 5 ) and lower serumconcentrations of cholesterol and HDL (FIG. 6 ).

C. comes Protection Against Metabolic Syndrome is AhR Dependent.

Supplementation of Coprococcus comes in AhR−/− mice was shown to be notsufficient to alleviate diet-induced metabolic impairments. Indeed, inAhR−/− mice, Coprococcus comes supplementation does not have any impacton body weight gain, glucose and insulin tolerance (FIG. 7 ) as well asfeatures of hepatic steatosis (FIG. 8 ). This lack of efficacy to treatmetabolic syndrome in HFD-fed AhR−/− mice demonstrates the AhR-dependentmechanism of C. comes.

1-22. (canceled)
 23. A method of treating a disease selected from thegroup consisting of metabolic syndrome and associated disorders, andinflammatory bowel diseases comprising administering a compositioncomprising a Coprococcus bacterium and/or a culture extract thereofexhibiting AhR agonist activity to a subject in need of treatment. 24.The method according to claim 23, wherein the bacterium is a Coprococcuscomes bacterium.
 25. The method according to claim 24, wherein thebacterium is Coprococcus comes strain deposited with the DeutscheSammlung von Mikroorganismen and Zellkulturen (DSMZ) under accessionnumber DSM 33359, or a mutant thereof.
 26. The method according to claim23, wherein the Coprococcus bacterium is a living Coprococcus bacterium.27. The method according to claim 23, wherein the culture extract is aculture supernatant.
 28. The method according to claim 27, wherein saidcomposition comprises Coprococcus culture supernatant.
 29. The methodaccording to claim 28, wherein said composition comprises a livingCoprococcus bacterium and a culture supernatant thereof.
 30. The methodaccording to claim 23, wherein the composition further comprises one orseveral additional bacterial probiotics capable of producing an AhRagonist that are selected from the group consisting of bacteriabelonging to the genera Allobaculum, Adlercreutzia, Anaerostipes,Bifidobacterium, Propionibacterium, Bacteroides, Eubacterium,Enterococcus, Ruminococcus and Faecalibacterium, Escherichia coli,Lactobacillus bacteria and Streptococcus bacteria.
 31. The methodaccording to claim 30, wherein said one or several additional bacterialprobiotics are selected from the group consisting of bacteria belongingto the genera Allobaculum, Adlercreutzia, Propionibacterium,Enterococcus, Escherichia coli, Lactobacillus bacteria and Streptococcusbacteria.
 32. The method according to claim 23, wherein the compositionfurther comprises one or several additional bacterial probioticsselected from the group consisting of strains available under CNCMdeposit numbers CNCM I-5019, CNCM I-5020, CNCM I-5021, CNCM I-5022 andCNCM I-5023, Ruminococcus gnavus ATCC 29149, Lactobacillus salivariusDSM 20555, Lactobacillus reuteri DSM 20016, Lactobacillus gasseri DSM20243, Faecalibacterium prausnitzii A2-165, Escherichia coli MG1665,Anaerostipes hadrus DSM 3319, Anaerostipes caccae DSM 14662,Anaerostipes butyraticus DSM 22094 and Allobaculum stercoricanis DSM13633.
 33. The method according to claim 23, wherein the compositiondoes not comprise any additional bacterium.
 34. The method according toclaim 23, wherein said composition is administered orally or rectally.35. The method according to claim 23, wherein the disease is metabolicsyndrome or associated disorders, said disorders being selected from thegroup consisting of cardiovascular diseases, insulin resistance, glucoseintolerance, type 2 diabetes, non-alcoholic fatty liver disease andlipodystrophy.
 36. The method according to claim 35, wherein thecardiovascular diseases are coronary heart disease, heart attack, orstroke.
 37. The method according to claim 23, wherein the disease ismetabolic syndrome or associated disorders, said disorders beingselected from the group consisting of cardiovascular diseases, insulinresistance, glucose intolerance, non-alcoholic fatty liver disease andlipodystrophy.
 38. The method according to claim 23, wherein the diseaseis metabolic syndrome.
 39. The method according to claim 23, wherein thedisease is an inflammatory bowel disease, Crohn's disease, ulcerativecolitis, indeterminate colitis (IC), other noninfective gastroenteritis,enteritis, enterocolitis, colitis, or pouchitis.
 40. The methodaccording to claim 39, wherein the disease is Crohn's disease orulcerative colitis.
 41. The method according to claim 23, wherein thedisease is not antibiotic associated colitis or Clostridium difficileassociated diarrhea.
 42. A Coprococcus comes strain deposited with theDeutsche Sammlung von Mikroorganismen and Zellkulturen (DSMZ) underaccession number DSM 33359, or a mutant thereof.